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Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, Pi, and pyruvate with AMP, PPi, and phosphoenolpyruvate (PEP) in three partial reactions as follows: 1) E-His + ATP →E-His-PP·AMP; 2) E-His-PP·AMP + Pi→ E-His-P·AMP·PPi; and 3)E-His-P + pyruvate → E·PEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosumPPDK (in the unbound state) reveals a three-domain structure consisting of consecutive N-terminal, central His-455, and C-terminal domains. The N-terminal and central His-455 domains catalyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide binding domain have been unsuccessful. The object of the present study is to demonstrate Mg(II) activation of catalysis at the ATP/Piactive site, to identify the residues at the ATP/Piactive site that contribute to catalysis, and to identify roles for these residues based on their positions within the active site scaffold. First, Mg(II) activation studies of catalysis of E+ ATP + Pi→E-P + AMP + PPipartial reaction were carried out using a truncation mutant (Tem533) in which the C-terminal domain is absent. The kinetics show that a minimum of 2 Mg(II) per active site is required for the reaction. The active site residues used for substrate/cofactor binding/activation were identified by site-directed mutagenesis. Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive; Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and Thr-253 and Gln-240 mutants were almost fully active. The participation of the nucleotide ribose 2′-OH and α-P in enzyme binding is indicated by the loss of productive binding seen with substrate analogs modified at these positions. The ATP, Pi, and Mg(II) ions were docked into the PPDK N-terminal domain crevice, in an orientation consistent with substrate/cofactor binding modes observed for other members of the ATP-Grasp fold enzyme superfamily and consistent with the structure-function data. On the basis of this docking model, the ATP polyphosphate moiety is oriented/activated for pyrophosphoryl transfer through interaction with Lys-22 (γ-P), Arg-92 (α-P), and the Gly-101 to Met-103 loop (γ-P) as well as with the Mg(II) cofactors. The Piis oriented/activated for partial reaction 2 through interaction with Arg-337 and a Mg(II) cofactor. The Mg(II) ions are bound through interaction with Asp-321, Glu-323, and Gln-335 and substrate. Residues Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of an active site loop, over the nucleotide ribose-binding site.

Potential domain-domain docking residues, identified from the x-ray structure of the Clostridium symbiosumapoPPDK, were replaced by site-directed mutagenesis. The steady-state and transient kinetic properties of the mutant enzymes were determined as a way of evaluating docking efficiency. PPDK mutants, in which one of two stringently conserved docking residues located on the N-terminal domain (Arg219and Glu271) was substituted, displayed largely unimpeded catalysis of the phosphoenolpyruvate partial reaction at the C-terminal domain, but significantly impaired catalysis (>104) of the ATP pyrophosphorylation of His455at the N-terminal domain. In contrast, alanine mutants of two potential docking residues located on the N-terminal domain (Ser262and Lys149), which are not conserved among the PPDKs, exhibited essentially normal catalytic turnover. Arg219and Glu271were thus proposed to play an important role in guiding the central domain and, hence, the catalytic His455into position for catalysis. Substitution of central domain residues Glu434/Glu437and Thr453, the respective docking partners of Arg219and Glu271, resulted in mutants impaired in catalysis at the ATP active site. The x-ray crystal structure of the apo-T453A PPDK mutant was determined to test for possible misalignment of residues at the N-terminal domain-central domain interface that might result from loss of the Thr453-Glu271binding interaction. With the exception of the mutation site, the structure of T453A PPDK was found to be identical to that of the wild-type enzyme. It is hypothesized that the two Glu271interfacial binding sites that remain in the T453A PPDK mutant, Thr453backbone NH and Met452backbone NH, are sufficient to stabilize the native conformation as observed in the crystalline state but may be less effective in populating the reactive conformation in solution.